Articular cartilage is avascular, aneural and contains no lymphatic vessels. It has a low level of metabolic activity compared with that of other connective tissues such as muscle but can be considered active for a cell that relies largely on glycolosis for energy. It also has an extensive extracellular matrix, which it relies upon to provide cartilage with its characteristic properties of low friction, pain-free articulation. The two main constituents of articular cartilage are the highly specialised chondrocytes, which are unique to cartilage and the matrix, composed of a complex, interconnecting arrangement of proteoglycans, collagens and non-collagenous proteins.
Articular cartilage can be divided into four main zones through its depth. These are the superficial; transitional; upper and lower radial; and calcified cartilage zones running from the outer articular surface to the deep subchondral bone, respectively. Although named zones are present, there are no ‘actual’ boundaries, which can be visualised between the zones. In each zone there are biomechanical and morphological variations (Dowthwaite at al, 2004), which include differences in cell morphology (size and shape), cell packing, metabolic activity and the thickness of the layers. Differences in matrix composition also exist between zones, with variations in the types and quantities of various collagens, proteoglycans, and non-collagenous proteins.
In spite of articular cartilage being only a few millimetres thick, it still manages to provide resistance to compression and displays the ability to distribute loads, thus in turn, reducing high stresses placed upon subchondral bone.
Chondrocytes
Normal articular cartilage contains one cell type, the highly specialised chondrocyte surrounded by extracellular matrix. In the majority of cases, the chondrocyte is “cytoplasmically isolated” (Archer and Francis-West, 2003) from its adjacent cells, seldom forming cell-cell contacts except in the most superficial part of the tissue. Each chondrocyte, therefore, is completely surrounded by matrix with which it freely interacts. Chondrocytes differ in their morphology and metabolic activities between the zones of articular cartilage. Generally the chondrocyte has a rounded or polygonal morphology, except at tissue boundaries where it may appear flattened or discoid, i.e. at the articular surface of joints (Archer and Francis-West, 2003). The principal role of the chondrocyte is in the maintenance of the intricate extracellular matrix of cartilage in particular the soluble, hydrophilic structures such as hyaluronan and aggrecan (Knudson, 2003). Intracellularly, the chondrocyte contains organelles that are typical of that of a metabolically active cell (Archer and Francis-West, 2003) that play a pivotal role in matrix synthesis, continually working to synthesise and turnover a large matrix to volume ratio, primarily composed of proteoglycans, glycosaminoglycans and collagens. Some chondrocytes also contain short processes or microvilli, which can detect mechanical alterations in the matrix. This is achieved as they extend from the cell directly into the matrix. Intracytoplastic filaments, lipid, glycogen and secretory vesicles enable chondrocytes to interact with the matrix. Mature chondrocytes are easily distinguished from other cells as they have a spheroidal morphology. They also have abundant amounts of type II collagen, large aggregating proteoglycans and specific non-collagenous proteins interwoven within a meshwork, which forms a cartilaginous matrix that covers and binds to their cell membranes.
Zones
Superficial Zone
The superficial zone (FIG. 6) is extremely thin and consists of two layers. The most superficial layer is acellular and consists of a thin, clear film of amorphous material known as the lamina splendens which overlies a sheet of fine, densely packed collagen type II microfibrils and comprises largely lubricin. The deeper cellular layer is composed of flattened, discoid chondrocytes enclosed within a collagen-rich matrix, which lie parallel to the articular surface (Dowthwaite et al, 2003). These cells synthesise matrix, which is abundant in collagen, fibronectin and water, and low in proteoglycans content compared to that of the deeper zones.
The dense layer of collagen fibrils have an orientation parallel to that of the surface and provide cartilage with its characteristic mechanical properties which include having high tensile strength and being able to resist shear force put upon it. The meshwork of collagen fibrils also permits the movement of molecules into and out of cartilage such as antibodies and large cartilage molecules, respectively.
Various studies have shown that the surface zone of articular cartilage is involved in the regulation of tissue development and growth. Developmental studies in our laboratory of Monodelphis domestica (South American opossum) have identified that articular cartilage grows by appositional growth from the articular surface (Hayes et al 2001) and that this method of growth allows for the distinct zonal architecture of this heterogeneous tissue to be established. These studies also showed that growth is driven by a slowly dividing population of chondrocytes in the surface zone of articular cartilage and a more rapidly dividing population of cells in the transitional zone (Hayes et al 2001). Not only do these observations account for the appositional nature of articular cartilage growth and zonal variation, they also suggest the presence of a specific articular chondrocyte progenitor cell population in the surface zone and a population of transit amplifying cells in the transitional zone.
Further, the surface zone has been found to be a signalling centre due to the expression of various growth factors and their receptors, which play a pivotal role in the morphogenesis of the diarthrodial joint via differential matrix synthesis (Dowthwaite et al, 2003). Recent in vitro studies have shown that the surface zone of bovine articular cartilage contains a progenitor cell population (Dowthwaite et al, 2004).
Acute articular cartilage injuries are a major concern for the athletic horse. They are a major cause of lameness and are associated with poor performance, early retirement and have a substantial negative economic impact. Chondral defects in the horse can occur through both disease and traumatic injury and if left untreated, leads to osteoarthritis characterised by progressive and permanent erosion of articular cartilage and associated bone and soft tissues of the joint.
Articular cartilage has a limited capacity for intrinsic repair. Partial thickness defects are non-healing and although full thickness defects may elicit an intrinsic healing response, the repair tissue formed is fibrocartilaginous and functionally inferior to the native tissue. Fibrocartilage is unsuitable as a replacement weight bearing surface and has been shown to undergo mechanical failure with use. Depending on the location and extent of the initial lesion, arthroscopic surgical debridement may be an effective treatment for returning a horse to athletic soundness. In many cases, however, additional techniques are needed to improve the healing response in the cartilage so as to preserve articular cartilage function.
A number of surgical treatments in the horse have been attempted both clinically and in experimental situations. Microfracture is a surgical procedure whereby the subchondral bone is perforated allowing mesenchymal stromal cells, blood cells and growth factors access to the chondral lesion. However, there is often poor integration, fibrocartilage formation and an aggrecan content that is not comparable to normal cartilage.
Cell based therapies are currently the treatment of choice for human articular cartilage repair but so far has limited use in the horse. Arguably, the current ‘gold standard’ for the biological repair of human chondral defects is autologous chondrocyte implantation (ACI). After monolayer expansion, the chondrocytes are placed within the chondral defect either by being injected through a periosteal flap that has been sutured over the lesion or by placing the cells on a collagenous membrane, which is secured within the lesion (matrix assisted chondrocyte implantation MACI). In either case, a limitation of the technique is that only relatively small lesions can be treated. When human chondrocytes undergo approximately 7 population doublings, they lose the ability to re-express the chondrogenic phenotype (in culture), thus limiting cell availability and this in turn is dependant on the amount of cartilage that can be harvested from the joint periphery which again is limited. However, the number of population doublings required for equine chondrocytes to lose chondrogenic potency has yet to be established. Successful repair of full thickness equine cartilage defects have been reported 12 and 18 months post-operatively using chondrocyte seeded scaffolds (Barnewitz et al., 2006, Frisbie et al., 2007). Articular chondrocytes and mesenchymal stromal cells (stem cells) are the two main cell sources used in cell-based cartilage repair therapies. It is unclear at this time however, if one cell type is more suitable than the other.
As mentioned above, others have previously isolated a population of progenitor cells from the surface zone of bovine articular cartilage (Dowthwaite et al., 2004). These cells were isolated using differential adhesion to fibronectin. They were able to form colonies from an initially low seeding density and were able to expand in culture without losing their chondrogenic phenotype. These cells were also engrafted into other connective tissue lineages and maintained the ability to form cartilage when transferred in to a 3D pellet culture system. Additionally, US patent 2006/0239980 teaches that articular cartilage obtained from the surface zone of human cartilage tissue can be enzymatically digested to produce a population of chondrocytes which, through culturing, can be dedifferentiated into chondroprogenitor tissue. However, there is no data in either of these documents concerning the phenotypic stability of this tissue and therefore the use of this bovine or human tissue as a reliable source of material for tissue repair is questionable.
Recently, we have extended the aforementioned studies and discovered, to our surprise, that it is possible to isolate a population of equine articular cartilage progenitor cells from the surface zone that have surprising and advantageous characteristics.
Here, equine articular cartilage progenitor cells (ACPC) and equine bone marrow-derived stromal cells (BMSC) are compared as potential cell sources for mammalian cartilage repair and, in particular equine cartilage repair. The study reports the isolation and partial characterisation of ACPC and compares their differentiation capacity to BMSC in vitro. We discovered that whilst ACPC and BMSC have functional equivalence in their multipotent differentiation capacity, to our surprise, chondrogenic induction of ACPC did not result in a hypertrophic cartilage phenotype, unlike BMSC. This means the ACPC retained the highly desirable hyaline cartilage phenotype. In contrast, after chonodrogenic induction BMSC exhibited a hypertrophic cartilage phentotype which is disadvantageous to any cartilage repair procedure as the cells can undergo terminal differentiation ultimately resulting in mineralisation of the matrix tissue, so producing stiffened joints. Therefore, equine ACPC have the highly desirable and, as yet, undemonstrated (in other cartilage stem/progenitor cells) phenotypic stability that makes them a reliable source of material for tissue repair.
Additionally, our novel equine progenitor cells exhibit phenotypic plasticity in that these cells can be functionally induced into various connective tissue types in order to produce different sorts of connective tissue.
It follows that our equine progenitor cells have significant use in cartilage repair. However, our progenitor cells could be used for the repair of other forms of connective tissue such as ligament, skin or bone.
Further, although our progenitor cells are suited to autologous repair, particularly cartilage repair, these cells also could be used allogeneically since many other stem cells have been shown to be immunosuppressive.